The major objective of this application is to better define the role of gamma/delta T-cells in the immune system. The principle goals are: (i) to extend our knowledge on the recognition requirements of gamma/delta TCR to better define gamma-delta TCR-ligand interactions; and ii) to characterize the ligands and responses of gamma/delta T-cells in both physiological and pathological situations. Five specific aims will be pursued. 1. Measurements of the affinity and kinetics of gamma/delta TCR binding to its ligand, and structural studies of gamma/delta TCR and TCR/ligand complexes. Soluble MHC ligands (class II E-alpha and E-beta chains and class I T10 and beta-2-microglobulin chains) have already been produced in E. coli. Several strategies will be used to generate soluble forms of TCRs that recognize these ligands, including truncation of the gamma and delta chain genes just before the transmembrane region, producing soluble gamma-delta TCR heterodimers from D. melanogaster cells (collaboration with Teyton), and preparing glycan-phosphatide-inositol-linked (gpi) TCR and expressing in CHO cells. Affinity will be measured using BIAcore technology. No information was provided concerning the proposed collaborative crystallographic analysis of gamma/delta TCRs and TCR/ligand complexes. 2. Characterization of T10/T22 and its recognition by gamma/delta T-cells. This will include analysis of a variety of surface markers on cells that express T10 from different tissues, thermal stability and turnover rate of T10, glycosylation pattern of surface T10, and possible association of other molecules with T10. These studies will be undertaken utilizing newly produced T10 monoclonal antibodies. 3. Evaluation of the gamma/delta T-cell contribution to the host defense against Yersinia pseudotuberculosis infection. A model system for studying gamma/delta T-cell involvement in host defense against Yersinia pseudotuberculosis infection will be more fully developed and characterized. Preliminary study has shown that 3-5 days after infection, a higher percentage of TCR-delta knockout mice have Yersinia in the spleen than normal mice. This suggests an active contribution of gamma/delta T-cells in containing the infection. In addition, the preliminary data show convergence in half of the normal mice of V-delta CDR3 length of a highly represented sequence of V-delta-4 positive IEL gamma/delta cells isolated 4 days after infection. This 'monoclonal' response in some of the mice suggests that gamma/delta T-cells are responding to the infection. Proposed study will monitor severity of the infection (count number of bacteria in various organs, determine survival rate, determine whether specific lesions develop, clearance time, kinetics of bacterial growth, etc.). 4. Identification and characterization of gamma/delta T-cells in Yersinia pseudotuberculosis infection. Based on study in Specific Aim 3, gamma/delta T-cells will be isolated from various organs and will be analyzed for: a) changes in gamma/delta T-cell populations and/or changes in cell surface marker expression; b) changes in gene expression using the new Affymetrix chip technology; and c) changes in gamma/delta TCR repertoire in response to infection (screen for usage of a particular V gene and/or CDR3 length). 5. Identification of targets of gamma/delta T-cells in a Yersinia infection. Ligands for gamma/delta T-cells responding to Yersinia will be characterized. This will include: a) identification of gamma/delta TCR sequences from single cells; b) sequencing of these genes, preparation of appropriate expression vectors, and transfection into recipient T-cells to regenerate TCR specificity; and c) utilization of the gamma/delta TCR transfectants to evaluate the nature of antigen and the requirements for antigen recognition. For the latter, ligands will be screened by using both pathogens and cells in the form of membrane associated as well as soluble fractions, assaying with fractionated protein and nonprotein extracts, and testing antigen alone or in conjunction with cells.